Urinary tract infection (UTI) is the most frequently diagnosed kidney and urological disease and E. coli is by far the most common etiologic agent. E. coil strains associated with cystitis and acute pyelonephritis often express secreted proteins and multiple fimbrial types which are thought to contribute to their ability to colonize the urinary tract, persist at this site, and elicit overt and recurrent disease. E. coil CFT073, a representative and highly virulent pyelonephritis strain, isolated by our group, will be used to study a secreted autotransported toxin, Sat, along with six other apparent autotransporter homologs; and the role in virulence of the invertible element-mediated phase variation of type 1 fimbrial gene expression. The specific aims of the study are: 1) to determine the mechanism of action and role in virulence of the Sat cytotoxin secreted by uropathogenic E. coli; and 2) to determine the contribution to virulence in the urinary tract of invertible element-mediated phase variation of type 1 fimbriae. We propose to characterize the Sat protein with respect to secretion, protease activity, internalization into the host cell, and toxin activity in vivo in the CBA mouse model of UTI. Six additional autotransporter genes have also been identified in the genomic sequence of strain CFT073 and will be characterized with respect to cytopathic or cytotoxic activities. With respect to type 1 fimbriae phase variation, we will construct locked-ON and locked-OFF mutants and assess the contribution to virulence of this critical adhesin both in the bladder and in the kidney. The long-term goal of this proposal is to understand the role of cytotoxins and adhesins in the virulence of uropathogenic E. coli in the urinary tract and to develop preventative strategies based on our understanding of the mechanism of this organism's pathogenesis.